1. Field of the Invention
The field of the present invention is transcobalamin receptor polypeptides and polynucleotides and the use of modulators thereof in the prevention, diagnosis, and treatment of cobalamin deficiency, tumors, and timmunological and inflammatory diseases and disorders.
2. Description of the Related Art
Vitamin B12 (cobalamin, Cbl) is a structurally complex water soluble molecule consisting of a planar corrin ring containing a central cobalt atom, a lower axial nucleotide (dimethylbenzimidazole) and an upper axial ligand which, in mammalian cells, is either a methyl, 5′ deoxyadenosyl or hydroxo group. Methyl-Cbl functions in the transfer of the methyl group from N5-methyltetrahydrofolate(methyl-FH4) to homocysteine in the de novo synthesis of methionine catalyzed by the enzyme methionine synthase (MS). 5′ deoxyadenosyl (ado-Cbl) is the cofactor for the rearrangement of methylmalonyl-CoA to succinyl-CoA, catalyzed by the enzyme methylmalonyl-CoA mutase (MMU) (14). The coenzyme function of hydroxo-Cbl is not known but it is an intermediary form in the interconversion of Cbl and is the substrate for the Cbl reductases in the neosynthesis of Cbl coenzymes.
The large size of Cbl (mol wt of CN-Cbl=1355), the complex three dimensional structure, and its hydrophilic properties prevent passive diffusion of this vitamin through cell membranes. A complex process has evolved in mammals requiring two carrier proteins and two membrane receptors for the assimilation of Cbl from the intestinal tract to its final destination in the tissue cells. Cbl released from food binds preferentially to a salivary R-protein favored by the acid pH in the stomach, and subsequently transfers to intrinsic factor (IF) at neutral pH in the jejunum as R-protein is digested by pancreatic trypsin. The IF-Cbl complex is carried to the distal ileum where it binds to specific receptors on the microvillous membrane. Recent reports have identified the IF receptor as cubilin, a 460 kDa membrane-associated protein that was purified from kidney and has been identified in the yolk sac and in the ileum. This protein binds IF-Cbl in the presence of Ca++ and interacts with amnionless a transmembrane protein involved in the endocytosis of cubilin bound IF-Cbl. The absorbed Cbl is released into the circulation bound to transcobalamin (TC), a Cbl binding plasma protein secreted by the vascular endothelium. Megalin, a multi-ligand binding protein expressed in the epithelial cells is involved in the reabsorption of TC in the kidney. The cDNA encoding cubilin has been cloned and the deduced amino acid sequence indicates that cubilin also belongs to the class of multi-ligand binding proteins.
The absorption of Cbl from the ileum is essential for maintaining Cbl homeostasis. Any perturbation of this process will ultimately lead to intracellular Cbl deficiency. Because of the fundamental role of Cbl in essential metabolic reactions, one of which is coupled to folate metabolism and, as a consequence, to nucleic acid synthesis, Cbl deficiency will lead to abnormal cell division and differentiation as observed in megaloblatic anemia resulting from both Cbl and folate deficiency. The neurological complications of Cbl deficiency are known to cause irreversible damage to the central as well as the peripheral nervous system (25).
Excluding dietary deficiency and secondary causes of malabsorption, such as tropical sprue and parasitic infestation, the primary causes of Cbl deficiency are, lack of IF as observed in patients with pernicious anemia, impaired release of Cbl from food, congenital IF deficiency and congenital TC deficiency. Cbl malabsorption and proteinuria characterize another disorder known as Imersiund-Grasbeck syndrome in which absorption of IF-Cbl in the distal ileum is affected by a defect in internalizing the receptor bound IF-Cbl.
The essential process of tissue distribution and cellular uptake of Cbl is mediated by TC, a plasma protein, and a membrane receptor (TCblR) for TC-Cbl that binds and internalizes TC-Cbl by endocytosis. TC is also required for the translocation of Cbl absorbed in the distal ileum and this function is impaired in congenital TC deficiency. The normal concentration of this protein in plasma is 0.4-1.2 pM, of which, approximately 10-30% is saturated with Cbl under conditions of normal Cbl homeostasis. Elevated levels of TC have been reported in certain autoimmune and myeloproliferative disorders. TC has been purified from human plasma and the cDNA encoding this protein has been isolated from endothelial cDNA libraries.
Reports that identified TC and TC mRNA in various tissues did not identify the specific cell in these tissues as the source of the protein. In addition, studies showing TC synthesis by liver cells, macrophages, fibroblasts, lymphocytes and ileal mucosa in culture do not provide information about the source of TC in vivo. Though all these cells may synthesize TC in culture, the finding that human umbilical vein endothelial cells (HUVEC) synthesize significantly more of this protein than other cells, and that the umbilical vein perfused ex vivo synthesizes TC, provides evidence that the vascular endothelium is the likely source of plasma TC. The extensive endothelial surface provides the capacity to maintain the circulating level of TC that has a relatively short half life of 60-120 min.
The Transcobalamin Receptor (TCblR)
The physiological process of Cbl uptake in cells requires a specific receptor on the cell surface that binds holo-TC with high affinity. Cbl binding proteins in serum have been identified and the role of TC in the cellular uptake of Cbl has been well characterized. Information on the cellular uptake of Cbl was derived initially from direct binding of Cbl to a membrane receptor in prokaryotes and from a more complex process in mammalian cells whereby a membrane receptor specifically binds TC-Cbl and internalizes the complex. The process is biphasic with an initial Ca++ dependent and temperature-independent binding of TC-Cbl to the receptor, followed by a slower second temperature-dependent step that translocates the vitamin into the cell and requires metabolic energy. The TC mediated uptake of Cbl has been identified in all mammalian cells studied and appears to be the only system for delivering Cbl into cells except for the liver, where uptake of Cbl bound to haptocorrin, another Cbl binding protein in the blood, occurs via the asialoglycoprotein receptor.
Studies in human skin fibroblasts established receptor-mediated endocytosis of TC-Cbl and the subsequent lysosomal degradation of TC to release the Cbl. Later studies showed that the receptors are expressed predominantly on membrane microvilli and the internalization of TC-Cbl occurs via clathrin-coated pits. Between 2000-6000 TC-Cbl receptors per cell are expressed during the proliferative phase of the cell and are down regulated to less than 300 receptors in non-dividing and fully differentiated cells. The increased requirement for intracellular Cbl during the early phase of cellular replication induces higher receptor expression in actively dividing cells that can account for a 10-30 fold increase in receptors in these cells. This expression of the TC receptor provides a unique target to selectively block Cbl uptake in cells that require the vitamin the most, i.e., actively dividing cells and to deliver antimetabolite analogs and Cbl-drug conjugates preferentially to rapidly proliferating cells.
Information on the structure of the TCblR protein is scant and results from different laboratories have not been consistent. The first attempt to solubilize TCblR was reported by Friedman et al. (J. Clin. Invest. 59:51-8, 1977), using placental membranes as the source of TCblR. They were able to show specific Ca++ dependent binding of TC-Cbl to placental membranes and to a Triton-soluble fraction of the membranes. Seligman and Allen in 1978 (J. Biol. Chem. 253:1766-72, 1978) reported the purification of the soluble receptor using conventional protein purification techniques coupled with affinity purification on a Sepharose-Cbl-rabbit TC column. They identified a major 460 kDa and a minor 40 kDa receptor by gel filtration chromatography and a 50 kDa protein by sucrose density gradient centrifugation. A major protein staining region corresponding to TC-Cbl binding activity in the gel was identified by non-denaturing PAGE but SDS-PAGE of the final product which can provide a better indication of purity and size was not reported. In their study, functional receptor activity was monitored using a mini DEAE ion exchange column to separate receptor bound TC-Cbl from free TC-Cbl. This method under the best conditions, only provides partial separation of the two fractions and an over estimate of TCblR activity (PI, personal observations) and therefore, the 2.9 n moles of TCblR activity recovered from 6 placentas is likely to be an over estimate. Based on the specific activity of the protein reported, and protein content, at best the final product would be 60% pure and therefore, amino acid and carbohydrate analyses reported cannot be accurate. Bose and Seetharam (Methods Enzymol. 281:281-9, 1997) used the procedure of Seligman and Allen with minor modifications to purified TCblR and, based on SDS-PAGE under reducing and non-reducing conditions, concluded that the purified TCblR migrates as a 72 kDa or 62 kDa protein respectively. They did not provide any data on the yield of functional activity, purity or specific activity of the final product. Their studies indicated that TCblR may exist as a non-covalent dimer in the lipid bilayer of the plasma membrane. They have not identified the primary structure and the gene encoding this protein. It is important to note that their results differed from earlier reports.
Studies to characterize TCblR from human placenta (Arch. Biochem. Biophys. 308:192-9, 1994) showed that the receptor is a 58 kDa protein with a core polypeptide of 41 kDa. Carbohydrate accounts for the remainder of the protein mass and is comprised of sialic acid (47%), and N-linked (24%) and O-linked (29%) sugars. These results were derived from enzymatic digestion of TCblR crosslinked to TC and changes in the molecular weight of the complex on SDS-PAGE. Taking into account all published data, complete characterization of the structure of TCblR had remained unresolved and the gene encoding this important receptor protein, unidentified.
Cbl in the Central Nervous System (CNS)
The neuropathological changes in the peripheral and central nervous system and the consequent functional abnormalities in Cbl deficiency provide compelling evidence in support of a role for this vitamin in maintaining a normal nervous system. However, clear evidence of Cbl deficiency in AD and other dementias is lacking. Among the biochemical and morphological changes observed in these disorders, hypomethylation of cellular components and apoptotic cell death have been observed in the brain. Such pathological changes can be caused by both Cbl and/or folate deficiency. The metabolic pathways involving these two vitamins have not been adequately characterized in brain tissue. The concentration of Cbl in the adult brain is ˜40-130 pg/mg tissue, a level similar to that in the spleen and kidney. The concentration of Cbl in most tissues including the brain is lower at birth and increases with age. In the fetal brain and liver, the Cbl requiring enzyme MS is highest during early embryogenesis and decreases as the fetus develops. The level of MeCbl in this tissue parallels the activity of the enzyme. Very little is known about TC and TCblR in the brain, and nothing is known about the expression of these essential proteins in aging and various brain disorders. The binding of TC-Cbl to brain tissue has been measured and appears to be higher in membranes prepared from the brain cortex than from the spinal cord. In addition, uptake of TC-Cbl has been reported in glial cells in culture.
TC is the major Cbl binding protein in cerebrospinal fluid (CSF) and the reported synthesis of this protein by brain cells would support the notion that the TC required for Cbl uptake may be synthesized in situ in the brain tissue. A role for Cbl and folate deficiency in various dementias could not be established by measuring total Cbl and folate levels in serum and CSF because they have provided ambiguous results. However, studies designed to evaluate methylation in the brain have identified altered methylation ratio of S-adenosylmethionine to S-adenosylhomocysteine (SAM/SAH) in the CSF. Hypomethylation in brain from Cbl deficient animals and in brain tissue obtained at autopsy from individuals with AD and dementia has been reported. These studies indicate that defects in the transmethylation pathways involving Cbl and folate may contribute to the pathogenesis of AD and dementias. In the absence of dietary deficiency, decreased cellular uptake may cause intracellular deficiency.
The TC-TCblR Pathway in Cancer Therapy
The recent advances in monoclonal antibody technology such as engineering antibody to eliminate most of the-immunogenic mouse protein component, or producing human antibody in a transgenic mouse, have significantly reduced some of the adverse effects of antibody therapy and have advanced the use of monoclonals to target specific antigens in cancer and autoimmune diseases. Preventing neovascularization of tumors with anti angiogenesis therapy, or by blocking the cellular uptake or intracellular metabolism of specific nutrients, are experimental approaches to suppress neoplastic growth. Because of the essential role of Cbl in recycling folate and thereby providing folate for DNA synthesis and maintaining intracellular SAM levels for methylation reactions, depleting intracellular Cbl would inhibit cellular replication. In humans, Cbl deficiency due to malabsorption or poor dietary intake takes several years to present clinically because of the large liver stores of Cbl. Infants born with congenital TC deficiency develop normally in the uterus because of the maternal supply of TC-Cbl. However they develop Cbl deficiency rapidly, i.e., after several months following birth because they lack TC. It has been difficult to produce an animal model of Cbl deficiency by Cbl depletion because of hepatic stores and contribution to the Cbl pool by the gut microflora. Some success was reported with the African fruit bats maintained in captivity for long periods on a Cbl deficient diet. It has been even more difficult to produce Cbl deficiency in vitro in cultured cells because of the TC-Cbl contribution from serum or serum factor supplements required for in vitro cultures. However, a carefully controlled study has shown that all cells have an absolute requirement for Cbl, and fail to replicate when intracellular Cbl decreases below a critical concentration. Evidence in support of the effects of Cbl deficiency on cell replication is also available from studies in which nitrous oxide (N2O) was administered for prolonged periods either to patients with acute leukemia to control their leukemia, or to patients following major surgery to alleviate their pain and these patients developed megaloblastic anemia akin to that seen in Cbl deficiency. N2O affects the reduced state of the cobalt in the Cbl molecule and this in turn affects the synthesis of methyl-Cbl the cofactor for the enzyme MS.
The strategy to inhibit cellular uptake of Cbl can utilize epitope specific monoclonal antibodies to the Cbl transporter, TC in blood and to the plasma membrane receptor, TCblR that facilitates the cellular uptake of TC-Cbl. mAbs to human TC have been generated (see Morgan et al., PCT Patent Publication No. WO 96/08515, published Mar. 21, 1996). Characterization of these murine mAbs has provided three distinct types of mAb, i.e., mAb that prevents the uptake of TC-Cbl by TCblR (receptor blocking), mAb that prevents the binding of Cbl to apo TC (Cbl blocking) and mAb that does not compromise the receptor binding and Cbl binding functions of TC (binding mAb). The receptor blocking and the Cbl blocking anti TC mAbs, either alone or in combination, could be used to prevent cellular uptake of Cbl. A similar effect could be produced with a mAb to TCblR that blocks the binding of TC-Cbl. These mAbs could serve as a biological modulator to arrest cellular proliferation by selectively depleting an essential nutrient. Studies with anti-TC mAbs show that these mAbs block Cbl uptake and inhibit cellular replication in vitro. The TC-Cbl binding mAb when coupled to drugs, toxins or radionuclides, could deliver these compounds to tumor cells via the TC-TCblR pathway. TCblR expression is increased during the proliferative phase of the cell cycle and in cancers, a large proportion of cells are actively dividing and therefore, this pathway could be exploited to deliver mAb-drug or Cbl-drug conjugates to replicating cells. Radioactive molecules or fluorescent compounds coupled to Cbl have been used to locate tumor mass by imaging techniques because more of these compounds accumulate in tumor cells compared to the surrounding tissue.
Clearly, there is a need in the art for additional methods of treating and preventing cobalamin deficiency, as well as cancer and other diseases related to cell proliferation, immune responses, and inflammation. The present invention meets this long-felt need by providing TCblR polypeptides and polynucleotides, which are useful in identifying modulators of CBL uptake and TCblR activity. In addition, the present invention provides novel TCblR modulators, including siRNA and antibodies specific for TCblR, and methods of making the same, which are used in therapeutic compositions to modulate Cbl uptake and treat cancer and other diseases associated with cell proliferation, immune response, and inflammation.